Environmental toxins and drugs often have clastogenic effects on living cells, i.e., cause microscopically visible damage or changes to cell chromosomes and cell cycle delays. Damage to cell chromosomes include breaks and rearrangements in the chromosomes, and changes in chromosome number. The clastogenic effects of a toxin or drug can lead to formation of micronuclei and presence of multiple nuclei in the living cells that are exposed to the toxin or drug. Therefore, micronucleus formation, and the presence of multi-nucleated cells can be used as indicators of genetic toxicity for certain environmental toxins and for evaluation of drug candidates during the development cycle of a new drug.
Evaluation of clastogenic effects of drugs or environmental toxins is usually accomplished by some form of micronucleus assay. A micronucleus assay is among a set of genetic toxicology assays wherein cultured cells are treated to induce formation of micronuclei and are then analyzed and scored for the extent of micronucleus induction. During a micronucleus assay, broken or detached chromosomes are separated from the spindle apparatus, and after cells undergo mitosis, the fragments become trapped in the cell cytoplasm and form micronuclei. The frequency of micronuclei formation in the micronucleus assay can then be measured and used to determine genotoxicity. In addition, the ratio of multi-nucleated cells to mono-nucleated cells provides information of cell-cycle delay, which is an early indicator of cytotoxicity.
An example micronucleus assay is the cytokinesis-block micronucleus (CBMN) assay for measuring micronuclei induction in cultured human and/or mammalian cells. During the CBMN assay, the cell cultures are exposed to the test substances; after exposure to the test substance, cytochalasin B for blocking cytokinesis is added; then the cell cultures are grown for a period sufficient to allow chromosomal damage to lead to the formation of micronuclei in bi- or multinucleated interphase cells, and then the cells are harvested and stained to permit micronuclei detection and scoring.
A micronucleus assay can be carried out in a multi-well plate. During such an assay, various fluorophores or fluorescent stains are applied to the cultured cells in the wells on the plate. Different stains are usually applied to cell nuclei and cytoplasm of the cultured cells. The nuclei stain and the cytoplasm stain are selectively excited using a different combination of excitation and emission filters, and separate fluorescent images of the stained cell nuclei and cytoplasm are obtained. Identification and counting of cells, nuclei, and micronuclei, in the micronuclei assay can be conducted manually by a technician or by an image processing system or software module based on the fluorescent images. Accurate detection of cell boundaries and correct assignment of nuclei and micronuclei to their respective cells are critical in scoring micronucleus induction that has occurred in the assay.
FIGS. 1A-1C are examples of fluorescent images of a cell sample stained with a cytoplasmic marker and a nuclear marker. In a two-channel fluorescent imaging system, the cytoplasm marker and the nuclear marker attached to the cells are selectively excited by light of two different frequencies, and their fluorescent emissions are separately captured in a cytoplasm image (as shown in FIG. 1A) and a nuclear image (as shown in FIG. 1B). The complete cell image is obtained by overlaying the two images together (as shown in FIG. 1C). In this example, the cytoplasm image can be processed to show the boundaries of the cells (i.e., red lines shown in FIG. 1A), and for calculating the number of nuclei per cell.
Current techniques for micronucleus scoring require images of both the cytoplasm and the nuclei for detecting cell boundaries and assigning nuclei and micronuclei to their respective cells. In essence, nuclei and micronuclei are assigned to their respective cells based on a calculation of where the cell boundaries are, principally because the cells are significantly larger than the nuclei and so the precision requirements for cell boundary detection are easier to satisfy. Hence, in the absence of a single stain that improves the contrast of both cytoplasm and nuclear material, a separate stain must be used to facilitate imaging of cytoplasm from that which is used to highlight the location of the nuclei. Such techniques are time-consuming because the samples must be stained and imaged twice, often require several trained personnel, and can be subjective in their determination. In particular, the requirement for separate cytoplasm imaging introduces significant amounts of time and cost for sample preparation and image processing.
Furthermore, the accuracy of cell boundary detection is significantly affected by the confluency state of the cell sample. High confluency makes cell boundary splitting very difficult and decreases the accuracy of these techniques. FIG. 2 shows an exemplary fluorescent image of a cell sample in a high confluency state. As cells are spaced more closely, proper assignment of nuclei to cells requires a higher precision of cell boundary detection. At the same time, cell boundary detection based on the cytoplasm image becomes more difficult with such high confluency.
Accordingly, there is a need for a fast, accurate, objective, automated, and cost effective method and/or system for the identification and quantification of micronuclei formation, and presence of multi-nuclear cells.